CN116751804A - VdCreA基因在大丽轮枝菌生长、致病力和碳代谢抑制中的应用 - Google Patents
VdCreA基因在大丽轮枝菌生长、致病力和碳代谢抑制中的应用 Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12N15/09—Recombinant DNA-technology
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- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- C07K—PEPTIDES
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- C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
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Abstract
本发明适用于功能基因技术领域,提供了VdCreA基因在大丽轮枝菌生长中的应用。提供了VdCreA基因在大丽轮枝菌致病力中的应用。提供了VdCreA基因在大丽轮枝菌碳代谢抑制中的应用。提供了一种棉花病害防治的药物,包括下调VdCreA基因或蛋白表达的试剂。本发明证明了VdCreA不仅在碳代谢抑制中具有重要作用,还影响大丽轮枝菌的生长发育和致病性,对大丽轮枝菌的防控和开发新型杀菌剂具有重要的理论价值,对促进棉花安全生产具有重要的现实意义。
Description
技术领域
本发明属于功能基因技术领域,尤其涉及VdCreA基因在大丽轮枝菌生长、致病力和碳代谢抑制中的应用。
背景技术
大丽轮枝菌(Verticilliumdahliae),属于半知菌亚门轮枝菌属真菌,含有很多降解植物细胞壁组分的降解酶(Cazymes),包括果胶酶类、纤维素酶类、淀粉酶类等,大丽轮枝菌通过与寄主的长期协同进化,根据寄主细胞壁的多糖成份不同而产生不同的碳水化合物水解酶,其功能赋予了大丽轮枝菌侵染植物特有的侵染特性,一些编码碳水化合物水解酶的基因直接影响大丽轮枝菌对植物的致病能力。碳代谢抑制(Carbon CataboliteRepression,CCR)作为全局调控因子,参与调控多个碳水化合物水解酶基因的表达,酿酒酵母、构巢曲霉等真菌中存在的碳代谢抑制参与致病菌的致病力,此外碳代谢抑制基因参与真菌的能量代谢、正常生长发育、碳水化合物代谢等细胞过程,目前已有研究表明,当破坏了碳水化合物水解酶相关基因,会直接影响大丽轮枝菌的致病性。
为了研究大丽轮枝菌碳代谢抑制基因的功能,通过检索大丽轮枝菌基因组数据库发现一个碳代谢抑制基因,基因编号为VDAG-00586,将其命名为VdCreA。
发明内容
本发明实施例的目的在于提供VdCreA基因在大丽轮枝菌生长中的应用,旨在解决上述背景技术中提出的问题。
本发明实施例是这样实现的,VdCreA基因在大丽轮枝菌生长中的应用。
优选地,所述生长包括生长速度、繁殖体产量。
优选地,所述繁殖体包括微菌核和分生孢子。
本发明实施例的又一目的在于提供VdCreA基因在大丽轮枝菌致病力中的应用。
优选地,所述致病力表现在以下指标中的一种或几种上:病情指数、菌丝穿透能力和寄主定殖能力。
本发明实施例的又一目的在于提供VdCreA基因在大丽轮枝菌碳代谢抑制中的应用。
优选地,VdCreA基因参与大丽轮枝菌由葡萄糖引起的碳代谢抑制。
本发明实施例的又一目的在于提供一种棉花病害防治的药物,包括下调VdCreA基因或蛋白表达的试剂。
本发明实施例对VdCreA基因在不同时间和不同组织中的表达进行分析,结果发现,野生型菌株V592在PDA固体培养基中培养12d、菌丝中VdCreA基因的表达量最高,根据同源重组原理,构建以VdCreA为靶标基因的敲除载体,通过农杆菌介导的遗传转化方法(ATMT)转化棉花黄萎病菌V592菌株的分生孢子,筛选获得2个VdCreA基因敲除突变体,同时构建以VdCreA基因为靶标的互补载体和过表达载体,筛选获得1个VdCreA互补体菌株和1个过表达体菌株,与野生型菌株V592和互补体菌株相比,VdCreA基因敲除突变体产生更多白色菌丝,生长速度减慢、产孢量下降、孢子变小且萌发率下降,对棉花的致病力降低;
碳源利用结果表明,VdCreA促进葡萄糖、半乳糖、蔗糖、棉子糖和木聚糖的利用,不影响对乳糖及果胶的代谢,对VdCreA基因进行碳代谢抑制试验,与野生型菌株V592相比,分别在纤维素和淀粉为碳源的培养基中加入葡萄糖后,VdCreA敲除突变体对纤维素和淀粉利用的抑制率明显降低,表明VdCreA基因参与大丽轮枝菌由葡萄糖引起的碳代谢抑制;
综上所述,VdCreA不仅在碳代谢抑制中具有重要作用,还影响大丽轮枝菌的生长发育和致病性。
附图说明
图1为实施例4提供的大丽轮枝菌ΔVdCreA突变株、互补菌株以及野生型菌株V592的菌落形态图;
图2为实施例5提供的大丽轮枝菌ΔVdCreA突变株、互补菌株以及野生型菌株V592的微菌核形成情况的显微观察结果;
图3为实施例6提供的大丽轮枝菌ΔVdCreA突变株、互补菌株以及野生型菌株V592的产孢量测定结果;
图4为实施例6提供的大丽轮枝菌ΔVdCreA突变株、互补菌株以及野生型菌株V592的产孢梗的显微观察结果;
图5为实施例7提供的大丽轮枝菌ΔVdCreA突变株、互补菌株以及野生型菌株V592对棉花的致病力测定(上图为致病力植株形态;下图为病情指数结果);
图6为实施例8提供的大丽轮枝菌ΔVdCreA突变株、互补菌株以及野生型菌株V592的菌丝对棉花的穿透能力测定结果;
图7为实施例9提供的ΔVdCreA基因突变体在不同碳源培养基上的表型、第9d的抑制率;
图8为实施例10提供的ΔVdCreA基因突变体菌株的淀粉酶和纤维素酶活性测定。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合附图及实施例,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。
在本发明实施例中,所述VdCreA基因的核苷酸序列如SEQ ID NO:1所示。
为了明确VdCreA基因敲除是否影响大丽轮枝菌分生孢子的形成,本发明实施例在野生型菌株V592为基础菌株构建了敲除VdCreA基因的大丽轮枝菌突变株ΔVdCreA,并在ΔVdCreA基础上进行基因互补和过表达,得到互补菌株ECVdCreA和过表达菌株OEVdCreA;所述敲除VdCreA基因的大丽轮枝菌突变株ΔVdCreA的构建方法利用同源重组原理完成,具体可参见现有技术获得敲除以及互补和过表达突变体(WANG S,XING H,HUA C,GUO H S,ZHANG J.An improved single-step cloning strategy simplifies the Agrobacteriumtumefaciens-mediated transformation(ATMT)-based gene-disruption method forVerticillium dahliae.Phytopathology,2016,106(6):645-652.);
本发明实施例分别以ΔVdCreA、互补菌株ECVdCreA以及过表达菌株OEVdCreA和野生型菌株V592为实验对象,进行一系列实验,结果表明:ΔVdCreA敲除体表现更多白色菌丝,生长速率减缓、孢子变小、产孢量及孢子萌发率均下降,致病性鉴定结果表明ΔVdCreA敲除体病情指数明显下降且病状明显减轻,说明VdCreA基因参与大丽轮枝菌的生长及致病过程;
通过外施葡萄糖观察碘液及刚果红分别染色淀粉及纤维素的情况,发现相较于野生型菌株,VdCreA基因敲除体在葡萄糖存在时,依然不影响对淀粉及纤维素的利用情况,说明VdCreA基因参与大丽轮枝菌中葡萄糖引起的碳代谢抑制反应;
碳代谢抑制是微生物代谢碳源时重要的生物过程,其中CreA调控多个与次级碳源代谢相关基因的表达,位于CCR途径的核心地位,CreA结构保守,是一类属C2H2类锌指DNA结合结构域的转录因子,经磷酸化和泛素化后结合到次级碳源相关代谢基因的启动子上,从而调控生物对次级碳源的利用,如纤维素酶、木聚糖酶及淀粉酶,这种复杂的CCR调控网络促进生物对复杂环境的适应性并增加其竞争能力,VdCreA突变体在不同碳源的生长情况间接反映了该基因对相应碳源代谢酶基因的表达情况,发现VdCreA敲除体在乳糖和果胶培养基中的抑制率与野生型无异,而在葡萄糖、半乳糖、蔗糖、棉子糖、木聚糖培养基中的抑制率明显高于野生型菌株,说明VdCreA基因不影响对乳糖、果胶代谢酶相关基因的表达,促进对葡萄糖、半乳糖、蔗糖、棉子糖、木聚糖代谢酶相关基因的表达;
本发明实施例通过对VdCreA基因进行了敲除及功能互补,并对该基因在大丽轮枝菌生长发育及碳代谢抑制中的功能进行了试验,证实了VdCreA基因在大丽轮枝菌的生长发育、产孢以及致病力等方面都发挥着重要的作用,并在识别并调控大丽轮枝菌的碳代谢抑制途径中具有重要作用。
以下结合具体实施例对本发明的具体实现进行详细描述。
实施例1、一种敲除VdCreA基因的大丽轮枝菌突变株ΔVdCreA的构建方法:
根据VdCreA基因(核苷酸序列如SEQ ID NO:1所示)上下游同源臂设计引物,构建敲除载体,以野生型菌株V592为初始菌株,构建突变株,获得敲除VdCreA基因的大丽轮枝菌突变株(ΔVdCreA-1,ΔVdCreA-2);
具体构建过程如下:
(1)目标基因同源臂的扩增:以VdCreA基因上下游为同源臂设计2对引物,以落叶型菌株V592的基因组DNA为模板,用I-5TM2×High-Fidelity Master Mix高保真DNA聚合酶扩增VdCreA基因的上下游同源臂,引物VdCreA-s-f和VdCreA-s-r扩增VdCreA基因的上游同源臂,引物VdCreA-x-f和VdCreA-x-r扩增VdCreA基因的下游同源臂;
PCR反应体系:I-5TM2×High-Fidelity Master Mix 25μL、VdCreA-s(x)-f/VdCreA-s(x)-r各1μL、及1μL的DNA加入到0.2mLPCR管中,用ddH2O补足至50μL体系,PCR反应程序为:98℃预热1min,98℃解链15s,60℃退火15s,72℃延伸15s,72℃保持5min,20℃维持2min结束反应,解链至延伸阶段进行30个循环;
取3~5μL PCR产物在加入Goldenview的1%琼脂糖凝胶电泳中进行凝胶电泳检测,于紫外凝胶成像仪下观察并拍照,将条带正确的剩余的PCR产物按照OMEGA生物公司产品Gel Extraction Kit步骤纯化目的基因片段并测定浓度;
其中上下游同源臂引物为:
VdCreA-s-f
CTTGCTGAGGTCTTAATTAACATCAGACATTCAGACCTCTTG(如SEQ ID NO:2所示)
VdCreA-s-r
AGTGCTGAGGCATTAATTAAGTGCGTCTGGGGTCCTGAGTTG(如SEQ ID NO:3所示)
VdCreA-x-f
CCCGCTGAGGACTTAATTAACCCTGCAGCAACATGTGTG(如SEQ ID NO:4所示)
VdCreA-x-r
CTCGCTGAGGGTTTAATTAAGGGGAAGACGAGGGATACGTAATG(如SEQ ID NO:5所示)
(2)本实施例所用敲除载体为pGKO-HPT,载体质粒用PacⅠ进行线性化酶切,体系如下:5μL的PacⅠ、35μL的载体质粒、5μL的1×Cutsmart,ddH2O补足至50μL,37℃水浴锅恒温酶切10-12h,次日取3~5μL PCR产物在加入Goldenview的1%琼脂糖凝胶电泳中进行凝胶电泳检测,于紫外凝胶成像仪下观察以检测酶切条带,小心切下目的片段用OMEGA生物公司产品Gel Extraction Kit步骤纯化线性化载体片段,再次凝胶电泳验证后将纯化载体片段分装并测定其浓度,将其保存于-20℃冰箱备用,避免反复冻融;
(3)In-fusion克隆:将扩增纯化后得到的目的基因上下游同源臂和线性化纯化后得到的载体片段用In-fusion酶进行连接,体系如下:目的基因上下游同源臂基因纯化产物各3μL、5×CE Multis Buffer和Exnase Multis各1μL,线性化载体2μL,将上述溶液置于37℃水浴锅中连接30min,置于冰上用于后续实验或暂存于-20℃冰箱,热击转化大肠杆菌并提取重组质粒;
(4)农杆菌介导的遗传转化:农杆菌的电击转化:从-20℃冰箱和-80℃冰箱中分别取出重组质粒和农杆菌感受态细胞迅速置于冰上解冻;取出1μL重组质粒加入到农杆菌感受态细胞中,冰置10min;擦干已灭过菌且预冷过的电击杯杯壁上的水份,加入混合液后放入电击仪中瞬时电击;加入500μL不含任何抗生素的LB液体培养基,移液器吹打混匀并吸取电击杯中的混合液,置于1.5mL离心管中,28℃摇床中200rpm摇培45min;瞬时离心,弃500μL上清液,用移液器充分混匀剩余菌液,在超净台中将菌液均匀涂布于含有Kan和Rif抗生素的LB固体培养基上,置于28℃恒温培养箱中暗培养2~3d,通过PCR筛选阳性转化子,将PCR正确的菌落摇菌获得农杆菌菌液。取100μL农杆菌菌液至10mL IMAS(含Kan)液体培养基中,28℃摇床中200rpm摇培至OD600值约为0.5,提前将收集好的大丽轮枝菌分生孢子放在冰上解冻,将大丽轮枝菌的分生孢子和农杆菌按照1:1的比例混合均匀;将200μL混合液均匀涂布于铺有灭过菌的IMAS固体培养基上,重复3次,仅涂布大丽轮枝菌分生孢子的平板作为阳性对照,而仅涂有农杆菌重组质粒的菌液的平板作为阴性对照;48h后,将滤纸从IMAS固体培养基上揭下并置于PDA(含HygB+Cef+Car+F2dU)平板上,26℃暗培养5~7d,待长出转化子,于超净工作台上挑取单个真菌菌落,划线接种于PDA(含Cef+HygB)培养基上,26℃暗培养10d左右,期间一直观察平板中真菌的生长情况,只有同源重组的敲除转化子会在该抗性平板上生长,而假阳性的转化子则不会生长,待阳性转化子长出,对其进行单孢分离并进行后续试验。最终获得两个敲除转化子。
实施例2、一种大丽轮枝菌VdCreA基因互补突变体EC-VdCreA的构建方法:
以实施例1构建的大丽轮枝菌突变株ΔVdCreA为初始菌株,获得大丽轮枝菌VdCreA基因互补突变体(ECVdCreA),所用的载体为p1300-Neo-oLiC-Cas9-TtrpC,用XbaI和BamHI双酶切线性化配置50μL XbaI和BamHI双酶切体系:p1300-Neo-oLiC-Cas9-TtrpC 20μL,XbaI 1μL,BamHI 1μL,1×Mbuffer 2.5μL,ddH2O25.5μL,37℃过夜酶切,次日切胶回收目的条带,使用II One Step Cloning Kit构建互补重组质粒,p1300-Neo-oLiC-Cas9-TtrpC与VdCreA基因用ExnaseII酶进行连接,p1300-NeO-LiC-Cas9-TtrpC的单片段连接体系体系如下:5×CEII buffer 2μL,p1300-Neo-oLiC-Cas9-TtrpC 200ng,目的基因片段80ng,Exnase II 1μL,ddH2O补足10μL,于37℃连接30min;
剩下的方法和敲除一致,与敲除体阳性转化子的获得的不同之处在于敲除突变体的获得是将大丽轮枝菌野生型V592的分生孢子1.0×106conidia/mL和含有敲除载体的农杆菌等比例混合,而互补突变体阳性转化子的获得是将VdCreA敲除突变体分生孢子1.0×106conidia/mL含有互补载体的农杆菌等比例混合;其次则是抗性筛选培养基的不同互补突变体阳性转化子的一筛培养基为PDA+HygB+Cef+Tim+G418,二筛培养基PDA+Cef+G418。
实施例3、一种大丽轮枝菌VdCreA基因过表达体OE-VdCreA的构建方法:
以大丽轮枝菌野生型V592为初始菌株,获得大丽轮枝菌VdCreA基因过表达体(OE-VdCreA),所用的载体与互补载体一致为p1300-Neo-oLiC-Cas9-TtrpC,不同之处在于过表达突变体阳性转化子的获得是将野生型V592的分生孢子1.0×106conidia/mL和含有过表达载体的农杆菌等比例混合,其它实验步骤不变。
实施例4、VdCreA基因在大丽轮枝菌生长中的应用:
(1)菌落生长速率的测定
以实施例1制备的敲除VdCreA基因的大丽轮枝菌突变株、实施例2制备的VdCreA基因互补突变体、实施例3制备VdCreA基因的过表达体以及野生型菌株V592为实验对象进行培养,分别取挑菌丝接种于PDA培养基中央,22℃黑暗培养,于接种后的第5天以及第9天,测量所有菌株的菌落直径,按照下式计算菌落的平均生长速率:
菌落的平均生长速率=菌落生长速度=(第9d平均菌落生长直径-第5d菌落平均生长直径)/4;
每个菌株设置3个重复,在第15天拍照记录菌落形态;
(2)菌丝的形态观察
大丽轮枝菌不同菌株在PDA平板上划线培养,再将灭菌的盖玻片斜插到划线部位,22℃暗培养3d,取出盖玻片在显微镜下观察菌丝生长情况,结果如图1所示,根据图1可以看出,VdCreA基因敲除体菌株的菌落平均生长速度明显低于野生型菌株,互补体菌株EC-VdCreA的菌落平均生长速度与野生型菌株无明显差异,过表达突变体菌株OE-VdCreA的菌落平均生长速度高于野生型菌株。说明VdCreA促进大丽轮枝菌的生长。
实施例5、微菌核量的测定:
以实施例1制备的敲除VdCreA基因的大丽轮枝菌突变株、实施例2制备的VdCreA基因互补突变体、实施例3制备VdCreA基因的过表达体以及野生型菌株V592为实验对象进行培养,得到菌饼,分别用打孔器对每种菌株打取10个左右菌饼接种于查氏(含kan)液体培养基中,26℃摇床中200r/min摇培3~5d;过滤收集分生孢子;调孢子浓度将分生孢子浓度调为1.0×106CFU/mL,吸取100μL均匀涂布于平铺玻璃纸的MM平板(NaNO32g、KH2PO41g、MgSO4·7H2O0.5g、KCl 0.5g,柠檬酸10mg,ZnSO4·7H2O 10mg、FeSO4·7H2O 10mg,NH4Fe(SO4)2)·12H2O 2.6mg,CuSO4·7H2O 0.5mg,NnSO4·H2O 0.1mg,H3BO30.1mg,Na2MoO4·2H2O 0.1mg,葡萄糖2g,1.5琼脂,加蒸馏水至1L。113℃,高压灭菌20min)上,22℃黑暗条件培养15d,拍照观察,刮取玻璃纸上的培养物并进行称重测量,并记录其湿重,再将微菌核在室温下放置48h晾干,在天平上称量,记录其干重数据;
如图2所示,根据图2可以看出,VdCreA基因敲除突变体在玻璃纸上不形成微菌核,在显微镜下也观察不到微菌核,而V592菌株和互补菌株在显微镜下均形成微菌核聚集体,因为微菌核是大丽轮枝菌生长侵染循环中的初侵染源,这表明VdILV6基因能降低大丽轮枝菌的侵染性能,从而降低其致病性。
实施例6、为了明确VdCreA基因敲除是否影响大丽轮枝菌分生孢子的形成,具体测定方法如下:
将浓度均为1.0×106CFU/mL的敲除突变体(实施例1构建所得菌株)、互补菌株(实施例2构建所得菌株)、过表达载体菌株(实施例3构建所得菌株)和V592分别接种到Czapek-Dox液体培养基中,在26℃、200r/min条件下摇培5d,分离孢子,将吸取1.0×106CFU/mL的孢子悬浮液100μL接种于查氏(含kan)培养基中,每个菌株设置3个生物学重复实验,在26℃摇床中200r/min速度振荡培养,期间每隔24h吸取1mL菌液,连续7d用血球计数板测量孢子浓度并记录数据;
结果如图3、图4所示,根据图3,从第3d开始,VdCreA敲除突变体菌株的产孢量明显低于野生型菌株V592,互补体菌株EC-VdCreA的产孢量与野生型菌株无明显差异,过表达突变体菌株OE-VdCreA的产孢量明显高于野生型菌株,说明VdCreA基因促进了大丽轮枝菌V592的产孢,根据图4可以看出,显微镜下观察突变体的产孢梗,ΔVdCreA突变体与V592菌株相比,菌丝体几乎不产生轮状分枝的产孢梗,互补菌株的产孢梗恢复至野生型菌株的水平。
实施例7、为了明确VdCreA基因敲除对大丽轮枝菌致病力的影响,以野生型菌株V592和互补菌株为对照,测定了VdCreA基因敲除突变体对棉花的致病力,致病性测定方法如下:
把上述构建的敲除突变体、互补菌株、过表达菌株和V592接种到Czapek-Dox液体培养基中26℃,200r/min摇培5d,棉苗第5片真叶长出后,通过使用根浸接种方法,每盆棉花接种200mL浓度为1.0×107CFU/mL的菌液,每个菌株重复3个水培盒(共36株棉苗),接种后每天都观察,从发病开始数病指,每隔3d数一次一般记录到发病后一个月,病害分级标准为:0级:不发病;1级:1~2片子叶发病;2级:1片真叶发病;3级:2片真叶发病;4级:3片及3片以上真叶发病,病情指数按照下式计算:
病情指数=[Σ各级病株数×级数/(总株数×最高病级)]×100;
病情指数为3次生物学实验重复平均值;
结果如图5所示,根据图5可以看出,相比于野生型菌株,接种VdCreA敲除体菌株的棉花延后5天表现叶片黄化、萎蔫及焦枯症状,在25d的发病症状明显低于野生型菌株,VdCreA敲除体菌株的病情指数也明显低于野生型和过表达体。
实施例8、为了分析VdCreA基因敲除导致大丽轮枝菌致病力下降是否与其穿透寄主的能力有关,以V592为对照,对各敲除突变体菌株进行了玻璃纸穿透实验,具体方法如下:
在倒好的MM基本培养基上铺己高温灭菌处理过的大小基本与培养皿一致的玻璃纸,用牙签挑取菌丝接种到至玻璃纸中央,分别培养3d将玻璃纸揭掉,让菌株再长7d,观察培养皿上是否能长出菌落,每个菌株设3个重复;
结果如图6所示,根据图6可以看出,VdCreA基因敲除突变体在接种第3d时能穿透玻璃纸,证明VdCreA基因敲除不会导致大丽轮枝菌在玻璃纸上的穿透能力丧失。
实施例9、大丽轮枝菌产生514个碳水化合物水解酶,其中152个与果胶、92个与纤维素、69个与木聚糖和61个与淀粉有关的碳水化合物酶,通过研究大丽轮枝菌VdCreA基因敲除突变体对不同碳源的利用情况,可间接反映VdCreA基因对不同碳源水解酶的调控作用,因此将各VdCreA突变体菌株和野生型菌株V592接种到不同碳源培养基上观察菌株的生长情况:
结果如图7所示,根据图7可以看出,在不同碳源培养基上培养第15d时,2个VdCreA基因敲除体菌株产生的黑色微菌核均明显少于野生型菌株V592,互补体菌株EC-VdCreA和过表达体菌株OE-VdCreA的黑色微菌核恢复;测量菌落直径进行抑制率分析,结果发现,与野生型菌株V592相比,在分别以葡萄糖、半乳糖、蔗糖、棉子糖和木聚糖为碳源的条件下,2个VdCreA基因敲除体菌株对真菌生长具有明显的抑制作用;在以乳糖、果胶为碳源的条件下,2个VdCreA基因敲除体菌株对真菌生长抑制作用与野生型菌株无明显差异,互补体菌株和过表达体菌株与野生型无异,说明VdCreA促进对葡萄糖、半乳糖、蔗糖、棉子糖、木聚糖的利用,不影响对乳糖、果胶的代谢。
实施例10、碳代谢抑制的首要任务是能敏感识别优选碳源,从而调控其他次级碳源,将VdCreA各突变体菌株和野生型菌株分别接种到以淀粉、淀粉+葡萄糖、纤维素、纤维素+葡萄糖为碳源的培养基上,用碘液和刚果红分别染色并测量其透明圈大小,计算葡萄糖存在条件下的抑制率;
结果如图8所示,在葡萄糖存在条件下,野生型菌株V592和VdCreA基因过表达体菌株丧失对淀粉及纤维素的利用,没有透明圈的产生,即没有淀粉和纤维素的分解,说明葡萄糖对淀粉和纤维素的利用具有抑制作用,抑制率均达到100%;VdCreA基因互补体菌株可产生较小的透明圈,表明葡萄糖对淀粉和纤维素有一定的抑制作用,抑制率分别达60.8%和59.1%;而2个VdCreA基因敲除突变体在葡萄糖存在时,依然可观察到明显的透明圈,说明VdCreA基因敲除解除了葡萄糖对淀粉和纤维素的抑制作用,反而促进了对淀粉和纤维素的利用,葡萄糖存在条件下对淀粉利用的抑制率均达-3.2%,葡萄糖存在条件下对纤维素利用的抑制率分别达-5.8%和-5.7%,以上结果表明,VdCreA基因在大丽轮枝菌中参与由葡萄糖引起的碳代谢抑制响应。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明的保护范围之内。
Claims (8)
1.VdCreA基因在大丽轮枝菌生长中的应用。
2.根据权利要求1所述的应用,其特征在于,所述生长包括生长速度、繁殖体产量。
3.根据权利要求2所述的应用,其特征在于,所述繁殖体包括微菌核和分生孢子。
4.VdCreA基因在大丽轮枝菌致病力中的应用。
5.根据权利要求4所述的应用,其特征在于,所述致病力表现在以下指标中的一种或几种上:病情指数、菌丝穿透能力和寄主定殖能力。
6.VdCreA基因在大丽轮枝菌碳代谢抑制中的应用。
7.根据权利要求6所述的应用,其特征在于,VdCreA基因参与大丽轮枝菌由葡萄糖引起的碳代谢抑制。
8.一种棉花病害防治的药物,其特征在于,包括下调VdCreA基因或蛋白表达的试剂。
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